One of the main difficulties in establishing whether or not there is a relationship between the degree of hyperglycemia and the long-term complications of diabetes is the lack of a reliable, objective method for assessing diabetic control. It recently has been suggested, however, that the measurement of glycosylated derivatives of normal hemoglobin, such as hemoglobin A.sub.1c, can be an indicator of such diabetic control. See, for example, D. Gonen and A. H. Rubenstein, Diabetologia, 15, 1 (1978). Such derivative of hemoglobin is formed by the post-transcriptional glycosylation of hemoglobin A.sub.0 (adult hemoglobin) at the terminal valine moiety of the beta chain. Such glycosylation is a slow chemical reaction which occurs throughout the life-span of the erythrocyte (about 120 days), the prevailing plasma glucose concentration being the most important factor governing the quantity of hemoglobin A.sub.1c formed.
Although a more rapid electrophoretic mobility of glycosylated hemoglobin was demonstrated by agar gel electrophoresis as early as 1957, quantitative data were not obtained by this method. The proportions of such hemoglobin only appeared to be much greater in some diabetic subjects than in others. Even those who first noticed glycosylated hemoglobin on the agar gel were not able to separate the glycosylated fraction to the extent that it could be quantified. S. Rahbar et al., Biochem. and Biophys. Res. Comm., 36, 838 (1969). Consequently, most of the quantitation studies have been performed by chromatography. D. W. Allen et al., J. Am. Chem. Soc., 80, 1628 (1958).
It since has been demonstrated by many investigators that there was indeed a quantitative importance of glycosylated hemoglobin determinations for the assessment of the status of sugar control in diabetic patients. However, an elaborate chromatographic separation was almost universally adopted. See, e.g., W. R. Holmquist and W. A. Schroeder, Biochem. Biophys. Acta, 82, 639 (1964) and L. A. Trivelli et al., N. Engl. J. Med., 281, 353 (1971). By such procedures, it was demonstrated that diabetic patients typically show a 2-fold increase in the level of hemoglobin A.sub.1c, the main component of glycosylated hemoglobins found in the peripheral blood.
The clinical importance of the glycosylated hemoglobin parameter in diabetic control and the relatively small span between normal and abnormal values calls for a rapid, routine assay of glycosylated hemoglobins which can be quantified with sufficient precision to reveal disturbances in sugar metabolism. Several modifications of minicolumn chromatographic procedures have been published and even introduced commercially for the quantitative assessment of glycosylated hemoglobins. Almost as many criticisms of these procedures appeared, citing the lack of precision. More laborious techniques such as isoelectric focusing and immuno techniques based on pure glycosylated hemoglobin antigenicity were suggested, but have not become accepted for routine clinical assays because of the sophisication of isoelectric focusing methodology and the difficulties in obtaining pure, specific antibody.